The plant hormone ethylene regulates a variety of morphological and physiological responses important for plant survival. The aims of this proposal serve to define the role of EIN6, which functions in an unresolved section of the ethylene signaling pathway. However, tools and approaches proposed in these experiments will have implications for many areas of plant biology and beyond. Preliminary sequence analysis reveals that EIN6 contains Jumonji domains and zinc-finger domains, suggesting a role as a chromatin remodeling enzyme. Specifically, EIN6 contains a Jumonji domain that is closely related to the JARID1 family of mammalian histone demethylases, which have recently been shown to demethylate Histone 3 Lysine 4 (an epigenetic mark associated with active transcription). Histone methylation is an area of particular interest given that this epigenetic modification is connected to cellular aberrations associated with cancer. We propose the use of chromatin immunoprecipitation (ChIP) coupled to target DNA identification through Solexa sequencing technology as an unbiased approach to identify EIN6 target sequences at the genomic level. Our proposed in vitro assay will determine if EIN6 does possess demethylase activity, which would be the first example of a histone demethylase in plants and add an interesting level of regulation to ethylene signaling. Our efforts to understand the mechanism by which EIN6 influences the ethylene response includes using a genetic approach to identify proteins that interact with EIN6 and a genetic screen in planta to identify enhancers of the ein6 phenotype. These studies will bring a new level of understanding to ethylene signal transduction and may reveal novel signaling components. With a more complete view of the pathway we may begin to apply our knowledge to agricultural and economic aspects of ethylene signaling, including fruit ripening and response to pathogen attack. The Specific Aims described here are centered on a key plant mutant that is insensitive to the critical growth hormone, ethylene. Defining the role of this previously uncharacterized gene will improve our current understanding of ethylene signaling in plants. This is an important step towards our long-term goal of applying our knowledge to agricultural and economic aspects of ethylene signaling, such as fruit ripening and response to pathogen attack. This protein of interest is predicted to function as a histone demethylase, which puts an intriguing spin on our work as this type of chromatin modification has not been previously described in plants and is linked to many human diseases including cancer. [unreadable] [unreadable] [unreadable]